This revised application is in response to the Program Announcement PA-09-119, Phased Innovation Award Program for AIDS Vaccine Research (R21/R33). This application proposes to produce neutralizing human monoclonal antibody (mAb) from human volunteers who received a polyvalent DNA prime-protein boost HIV vaccine in a Phase I clinical trial and their sera have demonstrated positive neutralizing activities against primary HIV-1 from clades A to E. If successful, this will be the first time that neutralizing human mAbs can be produced from HIV vaccinees. Detailed analyses of these mAbs will provide valuable information on their specificity, strength, and breadth of neutralizing activities. Similar to several available mAbs generated from HIV-1 infected patients, successful production of human mAb from HIV-1 vaccinees will provide useful information for the further optimization of Env-based HIV-1 vaccines. Specific Aims for R21 phase (Years 1~2): Specific Aim 1: To produce human hybridoma using PBMCs isolated from human vaccinees and to screen and identify Env-specific monoclonal antibodies including those with neutralizing activities. Specific Aim 2: To generate mAbs from single Env-specific B cells using a high throughput in vitro Ig expression strategy Specific Aims for R33 phase (Years 3-5): Specific Aim 3 To examine the strength and breadth of neutralizing mAbs and identify other protective functions of non-neutralizing mAbs produced from our proposed studies. Specific Aim 4: To analyze the epitope specificity of neutralizing mAbs produced in this proposal. Specific Aim 5: To expand the production of Env-specific mAbs from additional vaccinees including PBMCs collected at different time points in the immunization schedule. PUBLIC HEALTH RELEVANCE: We propose to use existing blood cells collected from human volunteers who received a novel HIV vaccine in an already completed clinical trial to generate a unique type of cells that can continuously produce highly specific antibodies - research tools that can guide the design of next generation of HIV vaccines.